A number of enveloped viruses, including retroviruses, hepatitis viruses, herpes viruses, orthomyxoviruses and paramyxoviruses, produce precursor envelope glycoproteins that require cleavage by a cellular dibasic amino acid processing endoprotease as one step in the process of envelope glycoprotein maturation. As precursor envelope glycoproteins are being synthesized, they are directed into the host cell secretory pathway for transport to the cell surface. As the precursor proteins move through the pathway, they are subjected to a variety of post-translational events including glycosylation and proteolytic cleavage (see, for example, Stein et al., 1990, J. Biol. Chem. 265, 2640-2649). The precursor human immunodeficiency virus (HIV) envelope protein gp160, for example, is co-translationally glycosylated and subsequently cleaved into gp120 and gp41 by a cellular dibasic amino acid processing endoprotease that apparently is localized in the Golgi apparatus. The gp120 and gp41 proteins are further glycosylated prior to reaching the infected cell surface. Cleavage of the HIV gp160 protein has been shown to be necessary for membrane fusion, syncytium formation and viral infectivity (see, for example, McCune et al., 1988, Cell 53, 55-67; Kowalski et al., 1987, Science 237, 1351-1355).
Although the genes encoding several dibasic amino acid processing endoproteases (also referred to as subtilisin-like protein convertases) have been isolated (see, for example, Barr, 1991, Cell 66, 1-3; Hakes et al., 1991, Endocrinology 129, 3053-3063; Kiefer et al., 1991, DNA and Cell Biology 10, 757-769; Lusson et al., 1993, Proc. Natl. Acad. Sci. USA 90, 6691-6695; Steiner et al., 1992, J. Biol. Chem. 267, 23435-23438, Nakagawa et al., 1993, J. Biochem. 113, 132-Nakagawa et al., 1993, FEBS Lett. 327, 165-171; Tsuji, et al., 1994, Biochem. Biophys. Res. Commun. 202, 1452-1459; Decroly et al., 1996, J. Biol. Chem. 271, 30442-30450), a number of cellular dibasic amino acid processing endoproteases remain to be identified, including CD4+ T-lymphocyte dibasic amino acid processing endoproteases responsible for cleaving the precursor envelope proteins of lentiviruses and lymphotropic viruses into envelope proteins, such as the enzyme that cleaves HIV gp160 into gp120 and gp41 in vivo. There is a need to identify cellular dibasic amino acid processing endoproteases that are responsible for in vivo cleavage of targeted substrates. Investigators have shown, for example, that the extent of proteolytic cleavage is a function of the sequence of amino acids at the dibasic amino acid processing site and of the dibasic amino acid processing endoprotease for hormones such as insulin and renin (see, for example, Oda et al., 1991, Biochem. Biophys. Res. Comm. 179, 1181-1186; Thim et al., 1986, Proc. Natl. Acad. Sci. USA 83, 6766-6770.
Nucleoside analogs are currently in use as antiviral drugs, particularly for treating retroviral infections as the analogs can inhibit the ability of the retroviral reverse transcriptase enzyme to make a DNA copy of the incoming viral RNA. For example, HIV infections are being treated with AZT (3'1-azidothymidine), ddI (2'3'-dideoxyinosine), ddC (2'3'-dideoxycytidine), and d4T (didehydrothymidine). Nucleoside analogs, however, have short half-lives and can exhibit substantial side effects. In addition, viruses often develop resistance to the nucleoside analog within a short period time of its administration.
Non-nucleoside inhibitors of HIV reverse transcriptase, such as TIBO (tetrahydro-imidazo(4,5,1-jk)(1,4)-benzodiazepin-2(1H)-one), BI-RG-587 (11-cyclopropyl-7-methyl-dipyrido-(2,3-b:3'3'-f)1,4-diazepin-6H-5-one), pyridones, and bis(heteroaryl)piperazines, are also being developed and tested. Since these compounds are highly selective for the HIV reverse transcriptase enzyme, they apparently cause less severe side effects than do nucleoside analogs. Decreased sensitivity of HIV to these agents, however, also develops rapidly.
The HIV-encoded aspartyl protease that processes the gag and gag/pol polyproteins to yield the mature structural proteins and enzymes required for virion formation (p24, p17, p15, reverse transcriptase) has also been targeted as an enzyme against which to design antiviral agents. HIV protease inhibitors, at least theoretically, can inhibit HIV production by chronically infected cells and, as such, have an advantage over reverse transcriptase inhibitors that apparently can only block replication if added to cells before HIV infection. Peptide-based substrate analogs are being prepared and tested. One persistent drawback of HIV protease inhibitors is the emergence of HIV strains that are resistant to the inhibitor being administered.
Other strategies for inhibiting HIV infection that are being pursued include inhibition of other HIV-encoded proteins such as Tat, Rev, and integrase; blocking entry of the virus into the cell by, for example, soluble CD4 receptor molecules; targeted delivery of toxins to HIV-infected cells; inhibition of viral functions using antisense technology; and immune constitution protocols. Although several of these technologies are at the early stages of development, clinical trials conducted using some of these technologies have been disappointing. For a recent review of present and future strategies to treat HIV infection, see Johnston et al., 1993, Science 260, 1286-1293.
Most assays used to test antiviral drugs are either in vitro or mammalian cell culture assays, many relying on the use of infectious virus. Mammalian cell culture assays are usually costly, complex, time-consuming, and potentially dangerous if infectious virus is used. Recently, a Drosophila cell-based assay was developed for screening inhibitors of the HIV Rev protein. For a review of methods to identify HIV inhibitors, see Johnston et al., ibid.
Thus, there remains a need to identify antiviral drugs with improved efficacy that have fewer side effects than known drugs and against which an infected host is less likely to develop resistance. A preferred class of inhibitors to identify are those that can be used to treat infectious diseases, such as HIV infections, in which proliferation of the infectious agent depends on dibasic amino acid processing endoprotease cleavage. In order to identify such drugs in a rapid and straightforward manner, an improved assay is required that is less complex, less expensive, less time-consuming, and more selective than currently used methods. There is also a need to identify CD4+ T-lymphocyte dibasic amino acid processing endoproteases, such as the enzyme that cleaves HIV gp160 in vivo, in order to identify specific inhibitors having greater selectivity and, hence, fewer side effects.